Correction: ERK1/2 Signaling Plays an Important Role in Topoisomerase II Poison-Induced G2/M Checkpoint Activation

[This corrects the article DOI: 10.1371/journal.pone.0050281.].

• The Control PARP and ETOP PARP panels are assembled from the same blot.Lanes 1 and 2 were used for the Control PARP panel, while lanes 1, 5, and 6 were used for the ETOP PARP panel.
• The Dox PARP panel was assembled from lanes 1, 3, and 4 of a different blot which was originally probed for PARP and then re-probed for Actin.The Control, DOX, and ETOP Actin panels are all assembled from this same blot.Lanes 1 and 2 were used for the Control Actin panel; lanes 1, 3, and 4 were used for the DOX Actin panel; and lanes 1, 5, and 6 were used for the ETOP Actin panel.
Actin was not used as a loading control in this experiment, but rather as an internal control that is expected to remain unaffected by the different treatments.
Here the authors provide a revised

Fig 1B In
Fig 1B In the originally published Fig 1B, in the left-hand DOX pERK1/2 and ERK 1/2 panels, an additional lane for time point 0.25 hr was removed between lanes 1 and 2. In the right-hand ETOP pERK 1/2 panel, the 2 hr band was removed in error, and lane 4 incorrectly shows the 3 hr band.Here the authors provide a corrected Fig 1.

Fig 2
Fig 2 In the originally published Fig 2, in the DOX panels of Fig 2A, lanes were removed from the original blot images; however, in each panel, the incorrect lane was removed, resulting in errors in lane labelling.In all panels of Fig 2B, a middle lane (the 1h time point) was removed, but the splice line was not marked between the first and third lanes (0h and 2h time points).Additionally, the

Fig 4A The
Fig 4A The western blot in the originally published Fig 4A is incorrect.Readers are referred to the revised version of Fig 6B for the correct results of this experiment.Here the authors provide a revised Fig 4 with the western blot panel of Fig 4A removed.

Fig 1 .Fig 6
Fig 1. DOX and ETOP induce G2/M arrest and ERK1/2 activation in MCF-7 breast cancer cells.(A) Log-phase MCF-7 cells were treated with DOX or ETOP at the indicated doses as described in Materials and Methods and incubated for 24 hr.The cells were analyzed for DNA content by FACS.Upper panel: histograms shown are cells treated with none, 1 µM DOX or 10 µM ETOP.Cell cycle phases are indicated.Lower panel: Graphs depict the percentage of cells with 4N-DNA content, indicative of G2/M phase of the cell cycle, and represent the mean ± s.d. of two sets of experiment with duplicate samples.(B) MCF-7 cells were incubated in the presence of 0.5 µM DOX or 10 µM ETOP for the hours indicated and analyzed for phospho-ERK1/2 and total-ERK1/2 by immunoblotting.https://doi.org/10.1371/journal.pone.0292423.g001

Fig 2 .
Fig 2. DOX and ETOP induce activation of Chk1 and Chk2 kinases and inhibition of Cdc2 kinase.(A) MCF-7Cells were incubated with 1 µM DOX (upper panel) or 10 µM ETOP (lower panel) for the indicated times for up to 2 hr.For the 4 hr and 6 hr time points, the cells were incubated for 2 hr with DOX or ETOP, washed with DMEM and incubated for additional 2 hr and 4 hr, respectively, in regular culture medium.Following treatment, Chk1 and Chk2 kinases were respectively immunoprecipitated from cell lysates and examined for kinase activity as described in Materials and Methods (Chk1 Activity and Chk2 Activity).Levels of Chk1 and Chk2 in the immunoprecipitates were determined by immunoblotting (Chk1 IP-WB and Chk2 IP-WB).*, as a negative control, kinase assay was carried out using immunoprecipitates obtained by incubating DOX-treated cell sample (6 hr time point) with non-immunized IgG.(B) Cells were treated as described above and incubated for 0, 1 and 2 hr.Cdc2 was immunoprecipitated from cell lysate and analyzed for levels of Cdc2-Tyr15 phosphorylation by immunoblotting (Cdc2-Tyr15).As a control, Cdc2 in the immunoprecipitates was assessed by immunoblotting (Cdc2).https://doi.org/10.1371/journal.pone.0292423.g002

Fig 4 .
Fig 4. Inhibition of ERK1/2 by specific siRNA diminishes topo II poison-induced G2/M arrest.(A) MCF-7 cells were transfected with ERK1/2 specific siRNA or control non-targeting siRNA and incubated for 2 days.The cells were then treated with 0.5 µM DOX or 5 µM ETOP, incubated for 24 hr and analyzed for DNA content by FACS.Left panel: histograms shown are DNA content analyses for the indicated cell samples.Right panel: bar graph depicts the percentage of cells in G2/M phase and presented as mean ± s.d. of three independent experiments in duplicate.** p<0.005 (n = 6), *** p<0.01 (n = 6), significant difference from cells transfected with control siRNA.(B) T47D cells were transfected with siRNA targeting ERK1/2 or control siRNA, incubated for 2 days and treated with 0.2 µM DOX or 5 µM ETOP.Left panel: levels of ERK1/2 in siRNA-transfected cells were determined by Western blotting.Right panel: the treated cell were incubated for additional 24 hr and analyzed for DNA content by FACS.Bar graph depicts the percentage of cells in G2/ M phase and presented as mean ± s.d. of two independent experiments in duplicate.** p<0.005 (n = 4), *** p<0.01 (n = 4), significant difference from cells transfected with control siRNA.https://doi.org/10.1371/journal.pone.0292423.g003

Fig 8
Fig 8 In the DOX and ETOP ERK 1/2 panels, lanes were removed to simplify the presentation.Additionally, in the ETOP ERK 1/2 panel, lanes 1-3 are incorrect and overlap with lanes 3 and 4 of the DOX ERK 1/2 panel, having been taken from the wrong part of the blot.Here the authors provide a corrected Fig 8, which contains all lanes on the original blots.

Fig 11
Fig 11Some bands in the originally published figure are duplicated across panels intentionally because of the experimental design.Specifically:•For the Control Caspase 8 and DOX Caspase 8 panels lanes 1 and 2 were used for the Control Caspase 8 panel, while lanes 1, 3, and 4 of the same blot were used for the DOX Caspase 8 panel.

Fig 8 .
Fig 8. Decrease of ATR level by shRNA had no effect on DOX-induced ERK1/2 activation.MCF-7 cells expressing ATR specific or control shRNA were treated with or without 1 µM DOX (upper panel) or 10 µM ETOP (lower panel) for 2 hr and analyzed for phospho-ERK1/2 (pERK1/2) and total ERK1/2 (ERK1/2) by immunoblotting.https://doi.org/10.1371/journal.pone.0292423.g005 Fig 11 in which each group (Control, DOX, and ETOP) is presented as an individual panel with a box around it, and the duplicated control lanes have been removed.S3 Fig Duplicates of lanes 1-2 of the DOX ERK 1/2 panel of Fig 6B and lanes 3-4 of the Actin panel of Fig 6B were reported in this figure in error.A corrected S3 Fig is provided in File S6.Additionally, the captions for S2 Fig and S3 Fig were incorrectly swapped during the publishing process.S2 Fig. Treatment with U0126 has no effect on the cell cycle profile of MCF-7 cells MCF-7 cells were incubated in the presence or absence of U0126 for 24 hr and analyzed for DNA content by FACS.Histograms shown are DNA content analyses for the indicated cell samples.S3 Fig.Transfection of non-targeting control siRNA had no effect on DOXinduced G2/M cell cycle arrest in MCF-7 cells MCF-7 cells were transfected with control non-targeting siRNA or left untransfected and incubated for 2 days.(A) Left panel: the cells were analyzed for protein levels of ERK1/2 and Actin by Western blotting.Right panel: Immunoblot densities of ERK1/2 and Actin were quantified using ImageJ software and relative ERK1/2 expression versus Actin determined.(B) The cells were then treated with 0.5 µM DOX, incubated for 24 hr and analyzed for DNA content by FACS.Histograms shown are DNA content analyses for the indicated cell samples.underlying 6C ETOP ATM activity blots are from a shorter exposure than that used for the figure.(ZIP) S4 File.Fig 8 Underlying Data.(ZIP) S5 File.Fig 11 Underlying Data.The underlying blot for the image shown in the original figure for the Control Caspase 8 and DOX Caspase 8 panels is not available; however, the authors provide an image of a shorter exposure of the same blot.Lanes 1 and 2 were used for the Control Caspase 8 panel, while lanes 1, 3, and 4 of the same blot were used for the DOX Caspase 8 panel.Additional Actin replicates are provided for the experiment shown in Fig 11B.One blot was probed first for the DOX PARP panel, and then re-probed for Actin.The blot showing PARP bands alone is not available, but the PARP bands remain visible on the blot after reprobing for Actin.(ZIP) S6 File.Revised Fig S3 and Underlying Data.(ZIP)